There are many procedures and processes that require the separation of blood into blood components or density graded layers. As used herein, “blood” may include peripheral blood, marrow blood or whole marrow. Known methods include density gradient additives which expand portions of the blood, discrete sample centrifugation, and continuous centrifugal separation. Separation of blood products may be required for diagnostic tests, blood donation, transfusions, or autogenic therapeutic reasons. The constituent components of a fractioned blood sample, listed from upper-most (least dense) to lower-most (highest density) fractions when a density separation is viewed from the side, are as follows: platelet-poor plasma, platelet-rich plasma, the “buffy coat” and red blood cells (RBCs). The buffy coat is the separated blood portion that contains the white blood cells, platelets, mesenchymal stem cells, hematopoietic stem cells. macrophages, adipocytes, osteoblasts, endothelial progenitor cells, very small embryonic like stem cells, blastomere like stem cells, and other nucleated cells.
Several techniques have been developed to isolate of the above noted fractions. Some of these techniques are substantially automated and performed by machine. Other techniques use substantially manual and feature centrifugation followed by some degree of manual post-processing to isolate the fractions. Alternative techniques involve manual isolation of centrifuged blood sample fractions using standard laboratory equipment and aseptic techniques. For example, as shown in FIG. 1 (Prior Art) a 50 cc or other appropriately sized conical tube is often used with centrifugation to fractionate a blood sample. There are benefits and shortcomings to each of the conventional blood fractionation methods. Some of the metrics used as indicators of the effectiveness of an isolation performance are as follows; time required to process blood, percentage of available fraction isolated, technician skill required to successfully produce quality isolations, and cross-contamination risk between isolated fractions.
As noted above, one blood fraction of interest is the buffy coat. The buffy coat is the separated portion of an un-coagulated blood sample that contains most of the nucleated cells, including but not limited to white blood cells, platelets, mesenchymal stem cells, hematopoietic stem cells, macrophages, adipocytes, osteoblasts, endothelial progenitor cells, very small embryonic like stem cells, blastomere like stem cells, and other nucleated cells after density gradient centrifugation. Typically, the buffy coat makes up less than 1% of the total volume of a blood sample. Although the buffy coat is predominately composed of white blood cells and platelets, the buffy coat also contains the various types of stem cells listed above. Stem cells, including mesenchymal stem cells (MSCs) are pluripotent blast or embryonic-like cells located in blood, bone marrow, dermis and perisosteum. In general these cells are capable of renewing themselves over extended periods of time as well as, under various environmental conditions, differentiating into cartilage, bone and other connective tissue. In this manner MSCs and other types of stem cells have been reported to have regenerative capabilities in a number of animal models.
Further, these finding are being extended in clinical trials to humans. Typical MSC therapies must be initiated with a source of autologous or non-autologous MSCs, and the proposed therapy can feature in vitro or in vivo MSC expansion. In view of the relatively tiny volume of the buffy coat and the relatively tiny quantity of MSCs in the buffy coat, it is useful to efficiently extract as much of the buffy coat from a sample as is possible in an aseptic and waste free manner.
The present invention is directed toward overcoming one or more of the problems discussed above.